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Detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in biological samples

a technology of methicillin-resistant and methicillin-sensitive staphylococcus aureus, which is applied in the field of pathogen detection, can solve the problems that detection of meca alone is not sufficient to identify mrsa directly from clinical samples, and mrsa represents a significant threat to public health, so as to increase the detection of target nucleic acids

Active Publication Date: 2011-02-15
QUEST DIAGNOSTICS INVESTMENTS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach enables accurate identification of MRSA and MSSA by producing distinct amplification products, effectively distinguishing between the two strains and reducing false positives from coagulase-negative Staphylococcal species.

Problems solved by technology

Accordingly, MRSA represents a significant threat to public health.
Therefore, detection of mecA alone is not sufficient to identify MRSA directly from clinical sample.

Method used

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  • Detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in biological samples
  • Detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in biological samples
  • Detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in biological samples

Examples

Experimental program
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Effect test

example 1

Conversion of Native Nucleic Acids to Sequence Modified Nucleic Acids

[0099]Genomic DNA was extracted from 6 nasal swab specimens using the QIAamp™ mini blood kit (Qiagen) extraction procedure with the ATL buffer modification. Nasal swab specimens were stored at −20° C. prior to extraction. Just prior to extraction, the swabs were thawed at room temperature for 20 minutes, 500 μL 1×PBS was added to the swab and the vial containing the swab followed by gentle agitation to mix. 450 μL of the suspension was then transferred to a 1.5 ml microcentrifuge tube and the suspension was centrifuged at 14,000 rpm for 10 min. The supernatant was carefully aspirated and the pellet was resuspended in 180 μL of Buffer ATL from the QIAamp™ mini blood kit. This kit was used to extract DNA according to the manufacturer's instructions. Eluted genomic DNA was split into two samples: one for conversion of nucleic acids to sequence modified nucleic acids (“converted DNA”) and the other was not converted (“...

example 2

Amplification of Sequence Modified Nucleic Acids

Top Strand Assays

[0101]To detect the sequence-modified top strand of integrated SCCmec, seven forward primers and two Scorpion™ oligonucleotides containing a reverse orfx primer and probe were designed. These primers detect the top strand of sequence modified integrated SCCmec MREJ (mec right extreme junction), types i & ii, iii, iv, v, and vii (Table 1). The forward primers were each specific to a single SCCmec type, but the Scorpion™ primer / probe, which hybridizes to orfx was not specific for any single SCCmec type. One Scorpion™ primer / probe and five forward primers are combined to detect sequence-modified SCCmec cassette types ii, iii, iv, v, and vii.

[0102]To determine the specificity of the primers shown in Table 1 for sequence-modified top strand of integrated SCCmec, samples of “converted DNA” or “unconverted DNA” were analyzed in singleplex reactions. A 10× Primer Mix was prepared using 0.1×TE pH 8 as the diluent. Final concent...

example 3

Multiplex Amplification and Detection of Sequence-Modified Nucleic Acids

[0116]A multiplex assay for sequence-modified spa, mecA, integrated SCCmec, and an internal control, was performed using the following Scorpion™ oligonucleotides and primers. A 10× Primer Mix was prepared using 0.1×TE pH 8 as the diluent. Final concentrations of each component in the 10× mix indicated in parenthesis: SA5-MSFTop1 (2 μM); SA5-MTopR1 (2 μM); SA1-MSFTop1 (2 μM); SA1-MTopR1 (2 μM); SA4-MSRTop2 (2 μM); SA4-MTopF1.1 (2 μM); SA4-MTop2.1 (2 μM); SA4-MTop3.1 (2 μM); SA4-MTopF4 (2 μM); SA4-MTopF5 (2 μM); IC-noloop-SFP-Dx2-DQ (1 μM); IC-SR4-Dx2 (1 μM). The reaction mix for a single well of a 96-well optical PCR plate included the following: 12.5 μL 2× Master Mix; 2.5 μL of 10× Primer Mix (components listed above); 4.0 μL Nuclease-free water; 1.0 μL Internal control amplicon; 5.0 μL bisulfite converted genomic DNA template (250 pg). The conditions for the real time PCR run on a Applied Biosystems AB7500 were...

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Abstract

Disclosed are methods of identifying a methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA) in a sample. The present invention provides a diagnostic method comprising modification of sequences of S. aureus by converting non-methylated cytosine residues ultimately into thymidine residues in the target nucleic acid. The invention further provides for the detection of modified sequences derived from the spa gene, the mecA gene, and the integrated SCCmec cassette of S. aureus.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 962,846, filed Jul. 31, 2007, hereby incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates generally to methods of pathogen detection. In particular, the present invention relates to methods of detecting methicillin-sensitive (MSSA) and / or methicillin-resistant Staphylococcus aureus (MRSA) in a biological sample.BACKGROUND OF THE INVENTION[0003]The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.[0004]Staphylococcus aureus (S. aureus) is a cause of a variety of conditions in humans, including skin infections (e.g. folliculitis, styes, cellulitis, impetigo, and furuneulosis), pneumonia, mastitis, phlebitis, meningitis, scalded skin syndrome, osteomyelitis, urinary tract infections, and fo...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/689C12Q2600/16
Inventor MCCARTHY, LARRYKONG, LILLYTABB, MICHELLELEE, MING-CHOU
Owner QUEST DIAGNOSTICS INVESTMENTS INC
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